Emergent mechanics of endocytic protein assembly

Max Ferrin

PhD candidate, Drubin/Barnes Lab

Clathrin-mediated endocytosis (CME)

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(Lacy et al., 2018)

CME is mechanically complex

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(Lacy et al., 2018)

Thesis outline: What emergent mechanics arise from interactions among endocytic components?

Chapter 1: Actin self-organization for load adaptation during CME

How is the force-generating endocytic actin network able to reorganize in response to increasing opposing force?

(Akamatsu et al., 2020)

Motivation: Size and shape of endocytic actin network changes as membrane tension increases

(Kaplan et al., 2022)

Theoretical approach: Agent-based model of minimal actin network in CME

(Nedelec and Foethke, 2007; Akamatsu et al., 2020)

Qualitative recapitulation of increased actin density in response to increased membrane tension

Analysis of simulations reveals quantitative measurements of actin self-organization

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Manipulation of actin-coat linkers abolishes adaptive response to tension by disrupting self-organization

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Model of load-responsive actin network self-organization

(Akamatsu et al., 2020)

Chapter 2: Type I myosin's actin-binding kinetics

Does the potential catch-bond activity of myosin serve an emergent function in CME?

Motivation: Diversity of myosin force sensitivity

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(Laakso et al., 2008; Pedersen, 2019)

Theoretical approach: Minimal model of CME with myosins

Generating a model non-processive motor

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Coarsely tuning force sensitivity to similar myosin measurements

(Greenberg et al., 2012)

Systematic quantitative analysis of CME simulations

Simulations predict optimal range of actin-binding kinetics for CME robustness

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Strong catch bonds hold actin in non-productive orientation

Weak catch bonds appear to exert force through actin network

Weak catch bond myosins qualitatively recapitulate internalization velocity measurements

(Manenschijn et al., 2019)

Summary: Weak myosin catch bonds aid CME robustness

Chapter 3: Actin network-mediated protein recruitment and membrane budding

How much of mechanically productive actin network architecture is encoded by its building blocks?

Experimental reconstitution approach: mix purified protein-coated SLBs with yeast cytoplasmic extract

Recapitulation of sequential endocytic protein assembly

Reconstitution of actin-mediated vesicle budding

Reconstitution of actin-mediated vesicle budding

Summary: CME actin network can self-organize for endocytosis-like force production

Chapter 4: Mechanical influence of liquid-like biomolecular condensate

Are capillary forces of a liquid condensate assistive or resistant to endocytic membrane bending?

Motivation: Mounting evidence of CME proteins forming liquid-like condensates

(Li et al., 2012; Bergeron-Sandoval et al., 2021; Day et al., 2021; Kozak and Kaksonen, 2022)

Motivation: Condensates can deform membranes through capillary forces

(Kusumaatmaja et al., 2021)

Capillary forces are present in CME

Mathematical model of CME mechanical energy

Condensate can provide net assistive force through wetting

Condensate surface tension results in energy barrier to initiation of membrane bending

The condensate can never be assistive to the flat-curved membrane transition

Droplet volume sets the energy barrier amplitude and position

Droplet volume sets the energy barrier amplitude and position

Summary: Condensate capillary forces inhibit membrane bending initiation, but assist completion

Manuscript figure outline

  1. Description of biological system and modeling approaches
  2. Qualitative features of toy model
  3. Validation of toy model results with continuum mechanics model
  4. Physiological significance of condensate mechanics by comparing model results to experimentally measured parameters

Fig. 1 panel sketches: Two complementary modeling approaches

Toy geometrical model Continuum membrane mechanics model
everything is a spherical cap discretized shapes governed by local forces
can be solved analytically must be simulated numerically
relatively easy to write out and program complicated math and simulation techniques required
unrealistic membrane shapes and kinks realistic membrane curvature
purely equilibrium solution can simulate temporal dynamics

Fig. 2: Qualitative features of toy model

Fig. 2 Supplement: Different behavior in constant coat area vs. constant curvature alternative models

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Fig. 2 Supplement: Free energy landscape of constant coat area vs. constant curvature alternative models

Fig. 3: Does model with realistic membrane bending recapitulate results of toy model?

Fig. 3 supplement: coated membrane stabilizes at predicted curvature

Fig. 3 supplement: condensate stabilizes at predicted contact angle

Fig. 3 results in progress: complementary simulations to toy model

Model condensate stalls initiation of membrane bending at similar parameter regimes between models

Fig. 4: what do condensate mechanics mean for CME function?

  • under which parameter regimes is there a large vs. negligible effect from the condensate?
  • which regime do experimental measurements place an endocytic condensate?
  • what parameters of the model might change over time in situ that could serve as regulatory features?

Fig. 4 panel: example analysis of condensate impact

Hypothetical fig. 4 panels: experimental estimation of condensate model parameters

(Brangwynne et al., 2009; Kozak and Kaksonen, 2022)

Hypothetical fig. 4 panel: regulatory potential of condensate mechanics

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Potential thesis timelines

  • if May 2023 filing date:
    • pause theory work
    • start writing thesis now
    • try to preempt reviewer-requested experiments for reconstitution manuscript
    • very quick attempt at minimal condensate fusion and FRAP experiments
    • finish condensate theoretical modeling project at Max Planck Institute in Dresden
  • if August 2023 filing date:
    • more buffer time for manuscript reviews
    • more time for optimizing condensate experimental system and data collection with summer undergraduate assistance
    • write my thesis over the summer
    • finish theoretical modeling in Dresden

Thank you!

References

Akamatsu, M. et al. (2020) “Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis,” Elife. Edited by P. Bassereau et al., 9, p. e49840. Available at: https://doi.org/10.7554/eLife.49840.
Bergeron-Sandoval, L.-P. et al. (2021) “Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling,” Proceedings of the national academy of sciences, 118(50). Available at: https://doi.org/10.1073/pnas.2113789118.
Brangwynne, C.P. et al. (2009) “Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation,” Science, 324(5935), pp. 1729–1732. Available at: https://doi.org/10.1126/science.1172046.
Day, K.J. et al. (2021) “Liquid-like protein interactions catalyse assembly of endocytic vesicles,” Nature cell biology, 23(4), pp. 366–376. Available at: https://doi.org/10.1038/s41556-021-00646-5.
Greenberg, M.J. et al. (2012) “Myosin IC generates power over a range of loads via a new tension-sensing mechanism,” Proceedings of the national academy of sciences, 109(37), pp. E2433–E2440. Available at: https://doi.org/10.1073/pnas.1207811109.
Kaplan, C. et al. (2022) “Load adaptation by endocytic actin networks,” Molecular biology of the cell, 33(6), p. ar50. Available at: https://doi.org/10.1091/mbc.E21-11-0589.
Kozak, M. and Kaksonen, M. (2022) “Condensation of Ede1 promotes the initiation of endocytosis,” Elife. Edited by M.I. Geli, 11, p. e72865. Available at: https://doi.org/10.7554/eLife.72865.
Kusumaatmaja, H. et al. (2021) “Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation,” Proceedings of the national academy of sciences, 118(36), p. e2024109118. Available at: https://doi.org/10.1073/pnas.2024109118.
Laakso, J.M. et al. (2008) “Myosin I can act as a molecular force sensor.,” Science (new york, n.y.), 321(5885), pp. 133–136. Available at: https://doi.org/10.1126/science.1159419.
Lacy, M.M. et al. (2018) “Molecular mechanisms of force production in clathrin-mediated endocytosis,” Febs letters, 592(21), pp. 3586–3605. Available at: https://doi.org/10.1002/1873-3468.13192.
Li, P. et al. (2012) “Phase transitions in the assembly of multivalent signalling proteins,” Nature, 483(7389), pp. 336–340. Available at: https://doi.org/10.1038/nature10879.
Manenschijn, H.E. et al. (2019) “Type-I myosins promote actin polymerization to drive membrane bending in endocytosis,” Elife. Edited by C.G. Burd, A. Akhmanova, and C.G. Burd, 8, p. e44215. Available at: https://doi.org/10.7554/eLife.44215.
Nedelec, F. and Foethke, D. (2007) “Collective Langevin dynamics of flexible cytoskeletal fibers,” New journal of physics, 9(11), p. 427. Available at: https://doi.org/10.1088/1367-2630/9/11/427.
Pedersen, R.T.A. (2019) Cooperative Force Generation by Actin Assembly and Myosin-I During Endocytosis, Proquest dissertations and theses. University of California, Berkeley.